During development, the trachea and esophagus are formed by separating a single foregut epithelial tube; when this fails an inappropriate connection or fistula of the trachea and esophagus is formed. Despite the common occurrence of congenital anomalies of foregut development, the pathways controlling this developmental process are incompletely understood and almost no knowledge exists regarding the molecular effectors of separation. Our preliminary data utilizing a novel mouse model indicate that the efnb2 gene may be a key regulator of this process. Efnb2 encodes ephrin-B2 a member of the Eph/ephrin family of cell signaling molecules that are involved in morphogenesis in a wide-variety of contexts. We will utilize mouse genetics to dissect the tissue specific requirement and cellular roles of ephrin-B2 in foregut morphogenesis and begin to determine where it fits in the genetic network controlling this process. These studies represent a new direction in which we will focus our understanding on the effectors of tracheoesophageal separation leading to improved understanding of structural birth defects of foregut development.